Assembly of the U4/U6 small nuclear ribonucleoprotein particle (snRNP) and its function in the spliceosome will be examined by genetic suppression analysis. Two different primary mutations will be used to probe different portions of the pre-mRNA splicing cycle. One mutation (a single-base substitution in U6 RNA) inhibits a conformational switch required for base-pairing of U4 and U6 RNAs. Over 100 independent spontaneous suppressors of the cold-sensitive growth phenotype associated with this mutation have been isolated. The suppressor strains are expected to harbor mutations in genes whose products participate in assembly of the U4/U6 snRNP or interact with U6 RNA in later steps of the splicing cycle. Characterization of the suppressor mutations will reveal the mechanism of U4/U6 snRNP assembly and will define interactions between U6 RNA and other splicing factors. The second primary mutation (a triple-base substitution in U4 RNA) results in the masking of essential sequences in U6 RNA, including the absolutely conserved "ACAGA box", during assembly and activation of the spliceosome. We have isolated 25 independent spontaneous suppressors of the cold-sensitive growth caused by this mutation. The suppressor strains are expected to harbor mutations in genes whose products interact with the U4/U6 snRNP during assembly and activation of the spliceosome. Their further characterization will reveal the mechanism of U4/U6 snRNP disassembly during activation of the spliceosome. The roles in splicing of the factors identified as suppressors of the two primary mutations will be further examined by genetic, biochemical, and physical analyses of the products of the suppressor alleles. The suppressor loci identified to date code for two spliceosomal RNAs (U4 and U6) and two splicing factors (Prp8 and Prp24). Several other loci have yet to be identified. The information obtained from the proposed study will significantly advance our understanding of a key step in eukaryotic gene expression. As a potential rate-limiting step in cell growth, splicing is almost certainly involved in the regulation of cell cycle progression. Indeed, certain mutations in the splicing factor Prp8, one of the subjects of this study, result in cell cycle arrest at the G1/S phase transition. Since loss of cell cycle regulation plays a major role in tumor progression, detailed knowledge of the splicing pathway is important for understanding carcinogenesis. Furthermore, the U4/U6 RNA interaction serves as a paradigm for the study of RNA dynamics, a new and important field of biochemical investigation.